Physical and functional probing of DEAD-box proteins as general RNA chaperones

描述（由申请人提供）：DExD/H-box蛋白质是结构化RNA进行的几乎所有过程所必需的，从前mRNA剪接和翻译到蛋白质和RNA的细胞内运输。这些蛋白质被认为使用来自ATP的能量来促进RNA构象变化和折叠转变，但在分子水平上对它们如何操纵RNA结构知之甚少。虽然DExD/H-box蛋白的许多RNA和RNA-蛋白靶标较大、复杂且难以在体外研究，但2002年显示粗糙脉孢菌CYT-19蛋白在几个线粒体I组内含子的折叠中起作用。进一步的研究表明，CYT-19也可以与II组内含子有效地相互作用，表明它具有一般的RNA伴侣活性，相关的酵母蛋白Mss 116 p显示出类似的功能。该项目的目标是使用CYT-19和I组RNA的明确定义的易处理系统来剖析DExD/H-box蛋白的RNA伴侣活性的机制。除了增加对基本细胞过程的了解外，这项工作对疾病也有影响，因为DExD/H-box蛋白是包括HCV在内的病毒复制所必需的，而人类DExD/H-box蛋白的过表达与结肠癌和前列腺癌有关。在目前的资助期间取得的进展导致了RNA分子伴侣活性的一般模型，围绕这一建议的中心。首先，CYT-19可以不加区别地破坏RNA结构，以依赖于RNA种类的相对稳定性而不是任何特定结构特征的效率展开非同源组I内含子的天然状态和长寿命的错误折叠构象异构体。然而，它的活性并不是完全不加选择的;它通过形成额外的“拴系”相互作用优先作用于结构化RNA，并且只有当P1不与RNA的其余部分“对接”成三级接触时，它才解开I组内含子（P1）的短螺旋。这些结果表明，CYT-19可能会优先破坏不能正确包装的错误折叠RNA。目前的建议的目标是深入研究RNA分子伴侣活性的物理基础DExD/H盒蛋白，探测特异性使用CYT-19及其生理底物，并测试假说的机制和影响的束缚和抑制三级接触。具体目标是：2）确定CYT-19是否以非同源RNA不存在的特异性作用于其同源I组内含子; 3）为了进一步探索三级接触形成的抑制作用，包括通过确定抑制是否存在于不同的结构元件和不同的I组内含子中来检验抑制是伴侣活性的一般特征的假设;和4）检验系链相互作用由CYT-19和相关DExD/H-box蛋白的高度碱性“尾”形成，并且这种相互作用在局部解旋期间可以保持完整的假设。预计结果将指导DExD/H盒蛋白在RNA代谢的各个方面的模型。 公共卫生相关性：该项目的目标是了解RNA伴侣蛋白如何帮助RNA折叠成特定结构并在结构之间交换。这些蛋白质是病毒复制所必需的，与人类癌症有关，因此了解它们的功能对于理解和最终治疗人类疾病至关重要。

DESCRIPTION (provided by applicant): DExD/H-box proteins are required for virtually every process carried out by structured RNAs, from pre-mRNA splicing and translation to intracellular trafficking of proteins and RNAs. These proteins are thought to use energy from ATP to facilitate RNA conformational changes and folding transitions, but relatively little is known on a molecular level about how they manipulate RNA structure. While many of the RNA and RNA-protein targets of DExD/H-box proteins are large, complex, and difficult to study in vitro, it was shown in 2002 that the Neurospora crassa CYT-19 protein functions in folding of several mitochondrial group I introns. Further work indicated that CYT-19 can also interact productively with group II introns, indicating that it possesses general RNA chaperone activity, and the related yeast protein Mss116p was shown to function similarly. The goal of this project has been to use the well-defined, tractable system of CYT-19 and group I RNAs to dissect the mechanisms of RNA chaperone activity by DExD/H-box proteins. In addition to increasing knowledge of essential cellular processes, this work has implications for diseases, as DExD/H-box proteins are required for replication of viruses including HCV, and overexpression of human DExD/H-box proteins is linked to colon and prostate cancer. Progress during the current funding period led to a general model for RNA chaperone activity, around which this proposal is centered. First, CYT-19 can disrupt RNA structure indiscriminately, unfolding both the native state and a long-lived misfolded conformer of a non-cognate group I intron with efficiencies that depend on the relative stabilities of the RNA species but not on any specific structural features. However, its activity is not fully indiscriminate; it acts preferentially on structured RNAs by forming an additional 'tethering' interaction, and it unwinds a short helix of a group I intron (P1) only when P1 does not 'dock' into tertiary contacts with the rest of the RNA. These results suggest that CYT-19 may disrupt preferentially misfolded RNAs that cannot pack correctly. The goals of the current proposal are to delve further into the physical basis of RNA chaperone activity by DExD/H-box proteins, to probe for specificity using CYT-19 and its physiological substrates, and to test hypotheses on the mechanisms and implications of tethering and inhibition by tertiary contacts. Specific Aims are: 1) to use an oligonucleotide displacement assay and directed hydroxyl radical footprinting to probe pathways of RNA chaperone activity on group I introns; 2) to determine whether CYT-19 acts on its cognate group I introns with specificity that is absent with a non-cognate RNA; 3) to probe further the inhibition by tertiary contact formation, including testing the hypothesis that the inhibition is a general feature of chaperone activity by determining whether it is present for different structural elements and within a different group I intron; and 4) to test the hypotheses that the tethering interaction is formed by a highly basic 'tail' of CYT-19 and related DExD/H-box proteins, and that this interaction can remain intact during local unwinding. Results are expected to guide models for DExD/H-box proteins in all aspects of RNA metabolism. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand how RNA chaperone proteins assist RNAs as they fold to specific structures and exchange between structures. These proteins are required for viral replication and are linked to human cancer, so understanding how they function is important for understanding and ultimately treating human disease.